PROJECT SUMMARY The overall goal of our program has been to improve the standard of care for brain tumor patients with a specific emphasis on glioblastoma multiforme (GBM). Towards this end, we initially developed oncolytic herpes simplex virus vectors (oHSV) to kill glioma cells, including the so-called glioblastoma stem cells (GSCs), and revealed the synergistic importance of viral oncolysis combined with immune stimulation (in situ oncolytic tumor vaccination). We demonstrated the advantage of blocking tumor-induced immune suppression and inducing anti-tumor immune responses during the process of in situ oncolytic tumor vaccination. Our studies of immune mechanisms demonstrated synergy of an oncolytic virus expressing a cytokine (IL12) with immune checkpoint inhibitors that forms an exciting avenue of research exploration for our next grant cycle, as well as for improved future clinical trials. Therefore, in this next grant cycle, we have designed studies to answer several important unmet needs in the immunovirotherapy of GBM: 1, How can a virus be better designed to stimulate an immune response to the tumor; 2, How can an oncolytic virus be used to overcome tumor-induced immunosuppression; and, 3, How can understanding tumor lymphocyte actions and macrophage reprogramming be utilized to improve therapeutic efficacy. We are taking advantage of this iterative process of oHSV development to continually improve outcomes. In the novel studies we have proposed, we are exploring the combined use of uniquely designed oHSV to overcome the current limitations of immune therapy by combined local intra-tumoral expression of co- stimulatory ligands in concert with cytokine stimulation of an in-situ oncolytic tumor vaccine response. This approach should eliminate distant tumor cells, while minimizing toxicity and be amenable to additional combinations. Our studies explore several hypotheses leading to improvement in vector design and efficacy. These studies will then be used to make novel oncolytic viruses incorporating the advantageous features learned from each of the aims in this grant proposal. We expect that this will lead to an improved immunovirotherapy approach to take into clinical trial for GBM. This strategy is not only novel and unique but we emphasize that it could save costs by shortening the length of treatment and also would apply to other tumors both in the brain and in the periphery.